Role of Phosphorylation Sites and the C 2 Domain in Regulation of Cytosolic Phospholipase A 2 Miguel

Cytosolic phospholipase A 2 (cPLA 2 ) mediates agonist-induced arachidonic acid release, the first step in eicosanoid production. cPLA 2 is regulated by phosphorylation and by calcium, which binds to a C2 domain and induces its translocation to membrane. The functional roles of phosphorylation sites and the C2 domain of cPLA 2 were investigated. In Sf9 insect cells expressing cPLA 2 , okadaic acid, and the calcium-mobilizing agonists A23187 and CryIC toxin induce arachidonic acid release and translocation of green fluorescent protein (GFP)-cPLA 2 to the nuclear envelope. cPLA 2 is phosphorylated on multiple sites in Sf9 cells; however, only S505 phosphorylation partially contributes to cPLA 2 activation. Although okadaic acid does not increase calcium, mutating the calcium-binding residues D43 and D93 prevents arachidonic acid release and translocation of cPLA 2 , demonstrating the requirement for a functional C2 domain. However, the D93N mutant is fully functional with A23187, whereas the D43N mutant is nearly inactive. The C2 domain of cPLA 2 linked to GFP translocates to the nuclear envelope with calcium-mobilizing agonists but not with okadaic acid. Consequently, the C2 domain is necessary and sufficient for translocation of cPLA 2 to the nuclear envelope when calcium is increased; however, it is required but not sufficient with okadaic acid.